Mako Nakaya

Foxp3 Inhibits RORγt-mediated IL-17A mRNA Transcription through Direct Interaction with RORγt

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.

Interleukin 27: a double‐edged sword for offense and defense

Cytokine‐mediated immunity plays a crucial role in the pathogenesis of various diseases including infection and autoimmune diseases. IL‐27, along with IL‐12, −23, and −35, belongs to the IL‐12 cytokine family. These family members play roles in regulation of Th cell differentiation. IL‐27 is unique in that although it induces Th1 differentiation, the same cytokine suppresses immune responses. In the absence of IL‐27‐mediated immunosuppression, hyperproduction of various proinflammatory cytokines concomitant with severe inflammation is observed. The immunosuppressive effects of IL‐27 depend on IL‐2 suppression, inhibition of Th17 development, and induction of IL‐10 production. Administration of IL‐27 suppresses some diseases of autoimmune or allergic origin, demonstrating its potential in therapy of diseases mediated by inflammatory cytokines. In this review, we discuss recent studies about the role of IL‐27 in immune regulation in view of its pro‐ and anti‐inflammatory properties and possible therapeutic application.

Cyclic Adenosine Monophosphate Suppresses the Transcription of Proinflammatory Cytokines via the Phosphorylated c-Fos Protein

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production from monocytic cells. Enhanced expression of interleukin-10 (IL-10) has been suggested to be the mechanism of suppression. However, cAMP is still capable of suppressing production of the cytokines TNF-alpha and IL-12 in IL-10-deficient dendritic cells (DCs). Here, we demonstrated that the transcription factor c-Fos was responsible for the cAMP-mediated suppression of inflammatory cytokine production. c-Fos accumulated at high amounts in response to cAMP and lipopolysaccharide (LPS). Overexpression of c-Fos suppressed LPS-induced cytokine production, whereas cAMP-mediated suppression of TNF-alpha and IL-12 was impaired in Fos(-/-) DCs or in RAW264.7 cells treated with c-Fos siRNA. c-Fos physically interacted with p65 protein and reduced the recruitment of p65 to the Tnf promoter. Multiple sites of c-Fos were phosphorylated by the IKKbeta protein. Thus, we propose that c-Fos is a substrate of IKKbeta and is responsible for the immunosuppressive effect of cAMP.

Effect of Estrogens on the Interferon-γ Producing Cell Population of Mouse Splenocytes

We examined the effect of 17β-estradiol (E2) on the cytokine production by mouse splenocytes. The production of interferon-γ (IFN-γ) was enhanced by E2 stimulation. E2 increased the number of cells expressing IFN-γ and the IFN-γ mRNA expression level in the cells. There were low- and high-level cells expressing IFN-γ in the population. The natural killer (NK) cells and NKT cells were the low-level cells expressing IFN-γ, and the number of these cells was increased by E2 stimulation. In addition, it was suggested that the enhancing effect of IFN-γ production by E2 was mediated through estrogen receptor (ER) α, and ERβ agonist stimulated IFN-γ production. ERs are expressed on plasma membrane as well as in nucleus. The ligand specific to plasma membrane-associated ER (mER) enhanced the IFN-γ production. In conclusion, our results indicated that E2 up-regulated the IFN-γ production by mediating ERα, ERβ and mERs.

Suppressor of cytokine signaling 1 protects mice against concanavalin A-induced hepatitis by inhibiting apoptosis

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling‐1 (SOCS1), which is a negative‐feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver‐specific SOCS1‐conditional‐knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)–induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild‐type (WT) and SOCS1‐deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun‐terminal kinase (JNK) activation, were enhanced in SOCS1‐deficient livers compared with those in WT livers. SOCS1‐deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti‐Fas antibody–induced apoptosis than were WT hepatocytes. Furthermore, SOCS1‐deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)‐α‐induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF‐α. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA‐induced liver injury. Conclusion: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis. (HEPATOLOGY 2008.)

Effect of royal jelly on bisphenol A-induced proliferation of human breast cancer cells

Royal jelly is known as a functional food containing many useful minerals. In this study, we found an anti-environmental estrogen activity of royal jelly. Bisphenol A (BPA) is an environmental estrogen that stimulates proliferation of human breast cancer MCF-7 cells. Royal jelly inhibited the growth-promoting effect of BPA on MCF-7 cells, even though it did not affect the proliferation of cells in the absence of BPA. In addition, this inhibiting effect of royal jelly was heat-stable.

A Major Lipid Raft Protein Raftlin Modulates T Cell Receptor Signaling and Enhances Th17-Mediated Autoimmune Responses

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

SOCS3 in T and NKT Cells Negatively Regulates Cytokine Production and Ameliorates ConA-Induced Hepatitis

Suppressor of cytokine signaling 3 (SOCS3), a negative-feedback molecule for cytokine signaling, has been implicated in protection against liver injury. Previous studies have shown that overexpression of SOCS3 in the liver by adenovirus or membrane permeable recombinant protein protected the liver from various injuries. However it remained uncertain in which type of cells SOCS3 suppresses liver injury. In this study, we demonstrated that forced expression of SOCS3 in T and NKT cells suppressed ConA-induced hepatitis using T and NKT cell-specific SOCS3 transgenic (Lck-SOCS3 Tg) mice. IFN-γ and IL-4 production was reduced in Lck-SOCS3 Tg mice as well as splenocytes treated with ConA. IFN-γ and IL-4 levels were also reduced in Lck-SOCS3 Tg mice administrated with α-galactosylceramide, suggesting that SOCS3 in NKT cells has suppressive function. Sustained expression of SOCS3 in an NKT cell line also resulted in reduced expression of various cytokines and transcription factors. In contrast, T and NKT cell-specific SOCS3 conditional knockout (Lck-SOCS3 cKO) mice were hypersensitive to ConA-mediated hepatitis. Isolated SOCS3-deficient NKT cells produced higher levels of IFN-γ and IL-4. These data indicate that SOCS3 plays a negative regulatory role in NKT cell activation and that forced expression of SOCS3 in NKT cells is effective in preventing hepatitis.

An ITAM-Syk-CARD9 signalling axis triggers contact hypersensitivity by stimulating IL-1 production in dendritic cells

A variety of reactive organic compounds, called haptens, can cause allergic contact dermatitis. However, the innate immune mechanisms by which haptens stimulate dendritic cells (DCs) to sensitize T cells remain unclear. Here we show that the coupling of ITAM-Syk-CARD9 signalling to interleukin-1 (IL-1) secretion in DCs is crucial for allergic sensitization to haptens. Both MyD88 and Caspase recruitment domain-containing protein 9 (CARD9) signalling are required for contact hypersensitivity (CHS). Naïve T cells require signals received through IL-1R1-MyD88 for effector differentiation, whereas DCs require CARD9 and spleen tyrosine kinase (Syk) signalling for hapten-induced IL-1α/β secretion and their ability to prime T cells. DC-specific deletion of CARD9, DAP12, Syk or NLRP3, but not MyD88, is sufficient to abolish CHS. All tested haptens, but not irritants, can induce Syk activation, leading to both the CARD9/BCL10-dependent pro-IL-1 synthesis (signal1) and reactive oxygen species-mediated NLRP3 inflammasome activation (signal2), required for IL-1 secretion. These data unveil an innate immune mechanism crucial for allergic contact sensitization to chemical compounds.

ESTROGENIC COMPOUNDS SUPPRESSED INTERFERON-GAMMA PRODUCTION IN MOUSE SPLENOCYTES THROUGH DIRECT CELL–CELL INTERACTION

SummaryHere, we reported the effects of 17β-estradiol (E2), isoflavone genistein (Gen), and daidzein (Dai) on the production of interferon (IFN)-γ by splenocytes isolated from C57BL/6N mice. When mouse splenocytes were stimulated with lipopolysaccharide, E2, Gen, and Dai suppressed the production of IFN-γ. However, when only nonadherent cell populations of splenocytes were tested, none of these estrogenic compounds suppressed IFN-γ production. This result indicates that IFN-γ production by nonadherent cell populations of splenocytes treated with estrogens is regulated by adherent cell populations. Moreover, direct cell-cell interaction between both populations was necessary for suppression of IFN-γ production by nonadherent populations. In addition, E2 conjugated with bovine serum albumin inhibited IFN-γ production as well as E2. This result suggests that the plasma membrane-associated estrogen receptor plays a prominent role in this suppression mechanism.